Identification Of Toxin Genes Of Corynespora Spp. Leaf Spot Pathogen In Tobacco And Determination Of Fungicide Toxicity In Guangx

Tobacco is an important economic crop,but the occurrence of leaf spot disease seriously restricts tobacco production.The leaf spot disease of tobacco caused by Corynespora cassiicola is a fungal disease,and its harm to tobacco production in Guangxi has been increasing in recent years,but there are still relatively few reports on the research of this disease.In order to provide a basis for effective control of tobacco Corynespora leaf spot disease in Guangxi,this study conducted research including isolation and identification of diseased leaf samples collected from major tobacco areas in Hezhou,Baise and Hechi,detection of pathogenic toxin protein subtypes,pathogenicity testing,indoor virulence testing of pathogen,screening of field control fungicide,and determination of the entire genome of pathogen containing toxin protein.The purpose of this study is to identify the species,toxin protein subtypes,and genomic information of Cas7 toxin subtype pathogenic bacteria of the pathogen causing tobacco Corynespora leaf spot in Guangxi,and to screen excellent fungicides for controlling tobacco Corynespora leaf spot.The main research results obtained are as follows:1.Pathogen of tobacco leaf spot disease collected from Guangxi tobacco region were isolated and purified,and their pathogenicity was determined using Koch’s postulate.Morphology,internal transcribed spacer（ITS）,and translation elongation factor（EF-1α）andβ-tubulin（TUB）primers were used for molecular identification.The results showed that all the 15 strains were pathogenic to tobacco.The positive surface of the bacterial colony on PDA medium was cyan gray,the hyphae were dense and villous,and the edge of the colony was gray.The spores are rod-shaped and slightly thinner at one end,and the molecular sequencing results have 99%or more homology with Corynespora cassiicola in Gen Bank.A phylogenetic tree was constructed by concatenation of three gene fragments,and the pathogen in this study converged with Corynespora cassiicola on a single branch.Based on the results of morphological and molecular biological analysis,it was identified that the pathogen of tobacco leaf spot caused by Corynespora cassiicola in Guangxi was Corynespora cassiicola2.Using specific primers for the toxin protein gene and toxin subtype determination for the pathogenicity of various host plants,the toxin protein subtype of Corynespora cassiicola in Guangxi was identified and analyzed for the first time.The results showed that the toxin protein subtype of 14 of the 15 strains of Corynespora cassiicola in Guangxi was Cas0,and the toxin protein subtype of 1 strain was Cas7.The results showed that there were many subtypes of Cas0gene,and the Cas7 toxin protein gene from the pathogen of tobacco Corynespora was first discovered in Tongde Township,Jingxi City.The results of pathogenicity testing showed that the two toxin subtypes of different pathogenic bacteria were pathogenic to tobacco（K326）,rubber（Haiken 6）,rubber（Haiken 1）,cucumber,balsam pear,tomato,and other crops,but not to banana（Jinfen 1 and Guijiao 6）.3.Using the method of mycelial growth rate,the effects of 12 fungicides on the biological activity of 8 strains of tobacco corynespora from Hezhou,Baise,and Hechi tobacco regions in Guangxi were measured.The partial sequence of the cytochrome b gene（Cytb）of Corynespora cassiicola was amplified and analyzed in order to explore the molecular mechanism of the decreased sensitivity of the pathogen to two types of fungicides such as QoIs.The results showed that the tested fungicides had different degrees of inhibition on the mycelial growth of Corynespora cassiicola,and the better inhibition effects were prochloraz,haloperidine,and pyrithiazide,with EC₅₀ values of 0.049±0.027,0.063±0.045,and 0.286±0.077 mg/L,respectively.The pathogen population has a high resistance to QoIs fungicides,and experiments have found that there is no mutation in the Cytb gene at the sites related to resistance in the Corynespora cassiicola.Field control experiments were conducted using four fungicides with good bacteriostatic effects in the indoor toxicity test and two commercially available microbial agents.The results showed that the tested agents had varying degrees of control effects on the disease,with pyrithiamide having a better control effect,followed by flupiramide,haloperidine,and prochloraz,with microbial agents having unsatisfactory control effects.4.In order to gain a deeper understanding of the Cas7 toxin subtype of Corynespora cassiicola,the second and third generation sequencing techniques were used to sequence,assemble,and predict the entire genome of the pathogenic fungus Corynespora multilocularis strain CBJT isolated from tobacco leaf spot in Guangxi.The assembled genome is 43.88 Mp in size and contains 171 Scaffolds,with a N50 of 1.68 Mp and a GC content of 51%.A total of14172 genes,39644 exons,14135 promoters,and 14109 terminators were predicted,with a repeat region length of 2.94 Mp and a repeat base ratio of 6.70%.The Cas7 gene sequence corresponds to positions 48951-49424 of the 46th Scaffolds in the entire genome sequence,clarifying the location of the Cas7 gene can lay the foundation for subsequent research on upstream and downstream regulation and influencing factors.The above information can provide data basis for the subsequent research on the functional analysis of important genes of Corynespora cassiicola and scientific control of tobacco leaf spot disease caused by Corynespora cassiicola.